Pasteurisation process for microbial cells and microbial oil

ABSTRACT

An improved pasteurisation protocol for pasteurising microbial cells is disclosed. The protocol has three stages, a first heating stage, a second plateau stage at which the cells are held at a (maximum and) constant temperature, and a third cooling stage. Both the heating and the cooling stages are rapid, with the temperature of the cells passing through 40 to 80° C. in no more than 30 minutes in the heating stage. The heating rate is at least 0.5° C./minute and during cooling is at least −0.5° C./minute. The plateau maximum temperature is from 70 to 85° C. By plotting the pasteurisation protocol on a time (t, minutes) versus temperature (T, ° C.) graph, one obtains a trapezium having an area less than 13,000° C. minute. Not only does this result in a smaller energy input (and so a reduction in costs), but a better quality (and less oxidised) oil results having a peroxide value (POV) of less than 1.5 and an anisidine value (AnV) of less than 1.0.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/248,065 filed Aug. 26, 2016; which is a continuation of U.S.application Ser. No. 14/311,461 filed Jun. 23, 2016, now U.S. Pat. No.9,457,108 issued Oct. 4, 2016; which is a continuation of U.S.application Ser. No. 13/467,843 filed May 9, 2012, now U.S. Pat. No.8,895,708 issued Nov. 25, 2014; which is a continuation of U.S.application Ser. No. 12/382,103 filed Mar. 9, 2009, now U.S. Pat. No.8,217,151 issued Jul. 10, 2012; which is a continuation of U.S.application Ser. No. 10/518,146, filed Dec. 16, 2004, now U.S. Pat. No.7,517,953 issued Apr. 14, 2009; which is a U.S. national stage under 35U.S.C. 371 of International Application No. PCT/EP2003/006553, filedJun. 20, 2003, which claims priority to EP Patent Application No.02258713.3 filed Dec. 18, 2002, and EP Patent Application No. 02254262.5filed Jun. 19, 2002, the entire contents of each of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a process for pasteurizing microbialcells, which comprises heating the cells at from 40° C. to 70° C. in nomore than 30 minutes. The rate of heating during the pasteurizingprocess can be at least 0.5° C./minute. The pasteurization process maycomprise three stages, namely a heating stage, a plateau (where thecells are held at constant temperature) and a heating stage. If onedepicts the pasteurization protocol graphically, the area under the time(minutes) versus temperature (° C.) graph is below 13,000° C. minute.After pasteurization, a polyunsaturated fatty acid (PUFA), such asarachidonic acid, or microbial oil may be extracted from the cells. Theoil may have a low peroxide value (POV) and/or low anisidine value(AnV).

Introduction

Polyunsaturated fatty acids, or PUFAs, are found naturally and a widevariety of different PUFAs are produced by different single cellorganisms (algae, fungi, etc). One particularly important PUFA isarachidonic acid (ARA) which is one of a number of Long ChainPoly-Unsaturated Fatty Acids (LC-PUFAs). Chemically, arachidonic acid iscis-5,8,11,14 eicosatetraenoic acid (20:4) and belongs to the (n−6)family of LC-PUFAs.

Arachidonic acid is a major precursor of a wide variety of biologicallyactive compounds, known collectively as eicosanoids, a group comprisingprostaglandins, thromboxanes and leukotrienes. Arachidonic acid is alsoone of the components of the lipid fraction of human breast milk and isthought to be essential for optimal neurological development in infants.Arachidonic acid has a wide variety of different applications includinguse in infant formula, foodstuffs and animal feeds.

WO-A-97/37032 (Gist-Brocades) refers to the preparation of a microbialPUFA-containing oil from pasteurized biomass. However, there is nodisclosure of rapid heating to, or cooling from, a temperature at whichpasteurization takes place.

Furthermore, no account is taken of the total amount of energy usedduring the pasteurization process.

WO-A-00/15045 and WO-A-01/67886 both refer to the use of Mucorales fungifor use in the preparation of foodstuffs. The first of these documentsrefers to the need to perform RNA reduction before including the cellsinto foods, and suggests using a heating step. A separate pasteurizationor heat shock can be performed. The second document suggests that aheating step to reduce RNA content may be avoided by allowing the fungalcells to be kept inside the fermenter vessel, and be allowed to “ripen”.

International patent application no. PCT/EP01/08902 refers to processfor preparing oil mixtures by combining a crude ω6 with a crude ω3PUFA-containing oil, to produce an oil mixture, and then purifying thecrude oil mixture.

Processes involving heating biomass, or microbial cells, are known. Itis also known, from WO-A-97/37032, that microbial cells can bepasteurized prior to extraction to a PUFA therefrom in the form of anoil. However, the present applicants have found that a newpasteurization process can improve the quality of that oil that can beextracted from the pasteurised cells. In particular, the resulting oilmay oxidise less, or be less oxidized, and may have a low peroxide value(POV) and/or anisidine (AnV). In addition, the applicants have foundthat this new pasteurisation process is more efficient because itrequires less energy. The process is therefore advantageous because notonly may it improve the quality of the oil, but it may reduce costssince less energy is required.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of temperature (° C.) against time (minutes) forpasteurisation at three pasteurisation protocols (A and C are within theinvention, B is provided for comparison);

FIG. 2 is a graph of temperature (° C.) against time (minutes) forpasteurisation at three different temperature plateaus (40, 70 and 85°C.); a graph of AnV against time (hours);

FIG. 3 is a graph of POV against time (hours);

FIG. 4 is a graph of AnV against time (hours);

FIG. 5 is a graph of POV (meg/kg) and AnV against temperature (° C.) forpasteurisation at two different (residence/plateau) times (8 and 300seconds);

FIG. 6 is a graph of temperature (° C.) against time (seconds) for twodifferent (residence/plateau) time (8 seconds for FIG. 6) at fivedifferent temperatures (60, 80, 100, 120, and 140 (° C.).

FIG. 7 is a graph of temperature (° C.) against time (seconds) for twodifferent residence/plateau) time (5 minutes for FIG. 7) at fivedifferent temperatures (60, 80, 100, 120, and 140 (° C.).

DESCRIPTION OF THE INVENTION

The present invention therefore provides an improved pasteurisationprocess of microbial cells. Despite requiring less energy, thepasteurisation process of the invention may allow for a better qualityproduct.

Thus, a first aspect of the present invention relates to a process forpasteurising microbial cells, the process comprising heating the cellsat (a temperature comprising) from 40° C. to (60° C. or) 70° C. in nomore than 30 minutes or heating the cells at a rate of at least 0.5°C./minute. This aspect therefore provides a rapid heating of themicrobial cells during pasteurisation, and such a high rate of heatingis not disclosed in the art. While the art gives pasteurisationtemperatures, there is no appreciation or discussion of the rate ofheating, or that this parameter would be important and that a relativelyrapid rate can provide benefits. Indeed, high heating rates arecounter-intuitive as they might be expected to cause oxidation orotherwise degrade the PUFA or oil that can be extracted from the cells.

A second aspect of the second invention relates to a process forpasteurizing microbial cells, the process comprising a pasteurisationprotocol that comprises (at least) three stages. These are, namely; a(first) heating stage, a (second) plateau stage (at which the microbialcells are held at a desired temperature, or where the cells aremaintained at a constant and/or maximum temperature), and a (third)cooling stage. This aspect of the invention is referred to as thethree-stage pasteurisation protocol. If this protocol is plotted on agraph of time versus temperature, a trapezium can result.

A third aspect of the invention relates to a process for pasteurizingmicrobial cells, the process comprising using a pasteurisation protocolsuch that the area under the time (minutes) versus temperature (° C.)graph is below 13,000° C. minute. The area under the time versustemperature graph gives the amount of energy expended in heating thecells during the pasteurisation process. It has been found that a rapidheating and/or rapid cooling (which correspond to the first and thirdstages of the second aspect, respectively) can provide advantages, suchas a better quality oil. In addition, the amount of energy required forthe pasteurisation process can be reduced in comparison withpasteurisation processes described in the art. This third aspecttherefore concerns to the energy input required for the pasteurisationprocess.

A fourth aspect of the invention relates to a process for pasteurisingmicrobial cells, the process comprising (heating the cells and so)maintaining the cells at an elevated temperature (T, ° C.) for a time(t, minutes), for example at a plateau stage, wherein the product tT(that is to say the multiplication of the time and temperatureParameters, for example during the plateau stage) is from 140 to100,800° C. minute. As will be realised, this fourth aspect is similarto the second aspect, in that it contains a plateau stage. The cellshere may be held at a constant or maximum temperature. The product tTcan therefore represent the area under the time versus temperature graphfor this plateau stage.

First Aspect—Rapid Heating

In this aspect the cells are heated so that the temperature of the cellspasses through or from 40° C. to 70° C. (or 60° C.) in no more than 30minutes (such as no more than 15 minutes). Preferably the time taken topass through from 40 to 70° C. takes no more than 40 to 50 minutes.Alternatively or in addition the cells are heated at a rate of at least0.5° C./minute. Of course, the microbial cells may start (or be heated)at a temperature below 40° C. For example, the cells may be at room orambient temperature. The cells may be at fermentation temperature, suchas 30° E 5° C. Thus the cells may be at from 20 to 40° C., such as from23 to 27° C. (or from 25 or 29 to 32 or 37° C.), when heating(pasteurisation) begins. In some cases the microbial cells may have beencooled, for example after fermentation has finished. Thus the cells mayhave a (starting) temperature of from 5 to 10° C., such as from 7 to 9°C., when heating begins.

The microbial cells may be heated so that their temperature rises above(60 or) 70° C. Thus, this may not be the final temperature of themicrobial cells during pasteurisation. Indeed, the cells maybe heated toa temperature above (60 or) 70° C. The temperature may rise until atemperature of from 70 to 90, 110 or 130° C., such as from 75 to 87° C.,and optimally from 78 to 84° C., is reached. The maximum temperatureduring pasteurisation may therefore be within these ranges, but for someembodiments may be up to 100, 120 or 140° C. Preferably, the cells areheld or maintained at that (maximum) temperature.

Hence it will be realised that the cells can be heated at a temperaturebelow, or starting from, 40° C., up to a temperature of 70° C. orhigher. The 40 to 70° C. range may provide a ‘snapshot’ in broaderheating/temperature range for which a time (and hence rate) can bespecified (and hence calculated).

It will be calculated that the heating (of 40 to 70° C. in 30 minutes)is a rate of 1° C./minute. However, the rate can be slightly lower thanthis if necessary, and in the first aspect the rapid heating means aheating rate greater than 0.5° C./minute. Preferably, the rate is atleast 0.6, 1.0, or even 1.5° C./minute. However, particularly fastheating rates are contemplated, depending on the equipment and thevolume or weight of the microbial cells to be heated. Heating rates inexcess of 2.0 or even 2.5° C./minute are thus within the invention.

Particularly high heating rates can be obtained using specialisedequipment. This may reach a high temperature in a short period of time,and in doing so this may minimise any oxidation or damage to the PUPA ormicrobial oil that may be later isolated. Thus, heating may take placeup to a maximum temperature of up to 140, 150 or even 160° C.Preferably, heating can be up to a temperature range within 100 to 180°C., such as 0.120 to 160° C., preferably from 130 to 150° C. Usingparticularly rapid heaters, these temperatures can be achievedparticularly quickly, for example within a time less than one minute (30seconds). Such temperatures could be reached within 20, 30, 40 or 50seconds, or may take up to 150, 175, 200, 225 or 250 seconds. However,such temperatures can be reached in as little as 2, 4, 6, 8 or 10seconds, for example if one is using an infusion heater, or withrelatively small samples. Thus, heating rates of up to 50, 100, 150 oreven 200° C. per minute are achievable. Slightly lower heating rates of5 or 10 to 50 or 60° C. per minute are thus possible, such as from 15 to45° C. per minute.

This rapid heating during rapid pasteurisation has been found not onlyto be more efficient, and requiring less energy, but it appears to be atleast one factor responsible for obtaining a better quality microbialoil (once extracted from the cells following pasteurisation).

Second Aspect—Three Stage Pasteurisation Protocol

The first stage can be a heating stage. This, in effect, corresponds tothe rapid heating described in the first aspect of the invention, andtherefore all features and characteristics of the first aspect apply tothe first (heating) stage of the second aspect mutatis mutandis.

The second stage is when the cells are at a plateau (in temperature).The cells can thus be held at a particular desired temperature (plus orminus 1 or 2, 5 or even 10° C.) for a desired length of time. The cellscan thus be maintained at a constant temperature. Preferably thistemperature (or range of temperatures), at the plateau stage, is themaximum temperature reached during the pasteurisation protocol. Thetemperature at the plateau stage (and/or the maximum temperature duringpasteurisation) is preferably at least 70° C. It may be below 90 or 100°C., suitably from 70 to 85° C., such as from 70 to 77° C. Alternatively,it may be from 80-160° C., such as from 100-140° C.

The length of time of the plateau stage, or at which the cells are heldat the desired or maximum temperature, can be from 5 seconds to 90minutes, such as from 1 or 10 to 80 minutes, for example from 20 to 70minutes. Optimally, this time is from 40 or 50 to 60 or 70 minutes, suchas from 45 to 65 minutes, advantageously at from 55 to 63 minutes,Particularly short times, e.g. from 8 seconds to 5 minutes are alsopossible.

The third stage is a cooling stage. Preferably, the cells are cooled toa temperature which is the same as, or within, the ranges mentioned forthe start of the heating (or first stage). Preferably, the microbialcells are cooled and/or heated linearly (in the first and/or thirdstages, as appropriate), that is to say when plotted on a time versustemperature graph the cooling or heating profile appears (approximately)as a straight line. The cells may be allowed to cool, or they may beactively cooled, for example using a heat exchanger and/or a coolingsubstance, for example (down) to ambient temperature or to roomtemperature, or lower.

Preferably the cooling rate is at least 0.4, 0.6, 1.0 or 1.5° C./minute.These values represent achievable cooling rates where the cells areallowed to cool. However, more rapid cooling rates are possible,especially if active cooling is employed. Thus, cooling rates of atleast 2.0, 2.5, 3.0 or even 3.5° C./minute are attainable. However,higher cooling rates, such as above 5° C. per minute are possible, e.g.from 7 or 10 to 50 or 60° C. per minute, preferably from 15 to 45° C.per minute.

The preferred heating and/or or cooling rate is preferably maintainedover at least 10, 20 or 30° C., although in some embodiments this can beachieved over at least a range of 40 or 50° C.

It will be realised that with a rapid heating stage and rapid coolingstage the amount of energy-used in pasteurisation can be reduced. Notonly can this result in cost savings, but it may not adversely affectthe quality of the (eventual) microbial oil, indeed it appears to havebeneficial effects on the oil.

Third Aspect—Area Under Time Versus Temperature Graph (Energy Input)

From the second aspect it will be apparent that if the pasteurisationprotocol of the invention is plotted on a time versus temperature graph,a trapezium shape is achieved. The first (heating) and third (cooling)stages may each be triangular in shape, while the middle or second(plateau) stage (the subject of the fourth aspect) is (usually)rectangular. The area under the time versus temperature graph representsthe amount of energy inputted into the system. By splitting thepasteurisation protocol into three parts, one can calculate the area ofthe graph, and therefore the energy input.

In the third aspect, the area under the time (in minutes) versustemperature (in ° C.) graph is below 13,000° C. minute. However, amountswell below this have been achieved, and values below 11,000, 10,000,9,000, 8,000 or even, 1,000° C. minute are possible. In preferredaspects of the invention, these values can be no more than 7,000, 6,000or 800° C. minute. In the graph referred to, the time is plotted on thex axis (or the horizontal axis or abscissa) and 0° C. represents theorigin. The temperature will thus be plotted on the y axis (or thevertical axis, or ordinate) and 0° C. represents the origin.

Once the microbial cells have been heated to their pasteurisationtemperature, they can then cool (or are cooled). The cells are usuallycooled to room or ambient temperature, or at least a temperature below30° C. There is therefore a time not only for the cells to be heatedfrom 30 to 60° C., but also a time for the cells to cool from 60° C.down to 30° C. One can sum these two times to provide a combined 30-60to 30° C. heating and cooling time. Preferably, this combined only isless than 150 minutes, such as less than 120 or 100 minutes. However,with smaller samples, much faster times can be achieved, and thecombined (30 to 60 and back down to 30° C.) time may be less than 70, 50or even 30 minutes.

Fourth Aspect—Pasteurisation Protocol with Plateau Stage

This protocol can be one according to the second aspect, where there isa (e.g. first) heating stage, and a (e.g. second) cooling stage, thesandwiching a (e.g. second, or middle or intermediate) plateau stage.However, that is not essential, and other pasteurisation protocols canbe envisaged. The fourth aspect relates to preferred features of thisplateau stage. All features and characteristics of the second (andother) aspects apply to this fourth aspect mutatis mutandis.

The cells are maintained or held at a particular desired temperature(plus or minus 1, 2, 5 or even 10° C.) for a temperature (T, ° C.) for atime (t, minutes). These two parameters can be multiplied together togive the product tT. This is suitably from 140 or 280 to 50,000 or100,800° C. minute. Preferably this product is from 500, 1,000, 2,000 or3,000 or even 6,000 up to 10,000, 18,000 or 25,000° C. minute.Optimally, the product tT is from 2,000 to 6,000, such as from 3,000 to5,000, optimally from 4,000 to 4,500° C. minute. In some embodiments,the product tT is from 13 to 900, such as from 100 or 200 to 700 or 800,optimally from 300 to 400 to 600 or 700° C. minute.

Thus in a similar manner to the third aspect, it will be realised thatthe product tT represents the area under the time versus temperaturegraph of the cells when maintained at the elevated temperature. Thus,the multiplication factor tT is, in effect, the area under the graph forjust the plateau (but not heating or cooling) stage.

Extraction of a PUPA

A fifth aspect of the present invention relates to a process forobtaining a PUPA from microbial cells, the process comprisingpasteurising the cells according to any of the first, second, third orfourth aspects of the invention, as previously described, and extractingand/or isolating a PUPA from the pasteurised cells.

A sixth aspect of the present invention relates to a microbial oil whichmay comprise at least 40% arachidonic acid (ARA) and/or may have atriglyceride content of at least 90%. The oil may have a POV of lessthan 2.5, 1.5, 0.8, 0.6 or even 0.5 and/or an AnV of less than 1.0. Theoil is preparable by the process of the fifth aspect.

Polyunsaturated Fatty Acids (PUFAs) and Microbial Oils

The PUPA can either be a single PUPA or two or more different PUFAs.

The or each PUPA can be of the n−3 or n−6 family. Preferably it is aC18, C20 or C22 PUPA. It may be a PUPA with at least 18 carbon atomsand/or at least 3 or 4 double bonds. The PUPA can be provided in theform of a free fatty acid, a salt, as a fatty acid ester (e.g. methyl orethyl ester), as a phospholipid and/or in the form of a mono-, di- ortriglyceride.

Suitable (n−3 and n−6) PUFAs include:

-   -   docosahexaenoic acid (DHA, 22:6 Ω3), suitably from algae or        fungi, such as the (dinoflagellate) Crypthecodinium or the        (fungus) Thraustochytrium;    -   γ-linolenic acid (GLA, 18:3 Ω6);    -   α-linolenic acid (ALA, 18:3 Ω3);    -   conjugated linoleic acid (octadecadienoic acid, CLA);    -   dihomo-γ-linolenic acid (DGLA, 20:3 Ω6);    -   arachidonic acid (ARA, 20:4 Ω6); and    -   eicosapentaenoic acid (EPA, 20:5 Ω23).

Preferred PUFAs include arachidonic acid (ARA), docosohexaenoic acid(DHA), eicosapentaenoic acid (EPA) and/or γ-linoleic acid (GLA). Inparticular, ARA is preferred.

The PUPA may be produced by the cells pasteurised in the process of theinvention, such as a microbial cell. This may be a bacteria, algae,fungus or yeast cell. Fungi are preferred, preferably of the orderMucorales, for example Mortierella, Phycomyces, Blakeslea, Aspergillus,Thraustochytrium, Pythium or Entomophthora. The preferred source of ARAis from Mortierella alpina, Blakeslea trispora, Aspergillus terreus orPythium insidiosum. Algae can be dinoflagellate and/or includePorphyridium, Nitszchia, or Crypthecodinium (e.g. Crypthecodiniumcohnii), Yeasts include those of the genus Pichia or Saccharomyces, suchas Pichia ciferii. Bacteria can be of the genus Propionibacterium. Themicrobial oil may be a liquid (at room temperature).

It is preferred that most of the PUFA is in the form of triglycerides.Thus, preferably at least 50%, such as at least 60%, or optimally atleast 70%, of the PUFA is in triglyceride form. However, the amount oftriglycerides may be higher, such as at least 85%, preferably at least90%, optimally at least 95% or 98% of the oil. Of these triglycerides,preferably at least 40%, such as at least 50%, and optimally at least60% of the PUPA is present at the α-position of the glycerol (present inthe triglyceride backbone), also known at the 1 or 3 position. It ispreferred that at least 20%, such as at least 30%, optimally at least40% of the PUFA is at the β(2) position.

The microbial oil may comprise at least 10, 35, 40 or 45% or more of adesired PUFA, such as arachidonic acid. It can have triglyceride contentof at least 90%. Preferably the microbial oil has a triglyceride contentof from 90 to 100%, such as at least 96%, preferably at least 98%, morepreferably at least 99% and optimally above 99.5%. Typically, themicrobial oil will have an eicosapentaenoic acid (EPA) content of below5%, preferably below 1% and more preferably below 0.5%. The oil may haveless than 5%, less than 2%, less than 1% of each of C₂₀, C_(20:3),C_(22:0) and/or C_(24:0) polyunsaturated fatty acid (PUFAs). The freefatty acid (FFA) content may be ≤0.4, 0.2 or 0.1. The oil may havelittle or no GLA and/or DGLA.

The microbial oil may be a crude oil. It may have been extracted fromthe cells by using a solvent, such as supercritical carbon dioxide,hexane or isopropanol.

Pasteurisation Process

Pasteurisation will usually take place after fermentation has finished.In a preferred embodiment, pasteurisation will finish the fermentation,because the heat during pasteurisation will kill the cells,Pasteurisation may therefore be performed on the fermentation broth (orthe cells in the liquid (aqueous) medium), although it can be performedon the microbial biomass obtained from the broth. In the former case,pasteurisation can take place while the microbial cells are still insidethe fermenter. Pasteurisation preferably takes place before any furtherprocessing of the microbial cells, for example granulation (e.g. byextrusion) crumbling, or kneading.

Preferably the pasteurisation protocol is sufficient to inhibit orinactivate one or more enzymes that can adversely affect or degrade aPUFA or microbial oil, for example a lipase.

Once fermentation has been finished, the fermentation broth may befiltered, or otherwise treated to remove water or aqueous liquid. Afterwater removal, one may obtain a biomass “cake”. If pasteurisation hasnot taken place, then the dewatered cells (or biomass cake) can besubjected to pasteurisation.

PUFA Extraction Process

The PUFA (or microbial oil, usually comprising the PUPA) may then beextracted from the (pasteurised) microbial cells. Preferably, it isextracted from (e.g. dried) granules (e.g. extrudates) containing thecells. The extraction can be performed using a solvent. Preferably anon-polar solvent is used, for example a C₁₋₈, preferably C₂₋₆, alkane,for example hexane. One may use carbon dioxide (in a liquid form, forexample in a super critical state).

Preferably, the solvent is allowed to percolate over the dried granules.Suitable micro-organism granulation and extrusion techniques andsubsequent extraction of a microbial PUPA containing oil, are describedin WO-A-97/37032.

The solvent allows one to obtain a crude PUFA containing oil. This oilcan be used in that state, without further processing, or it can besubjected to one or more refining steps. However, a crude oil is usuallyone that contains a solvent, such as a solvent used to extract the oil(e.g. hexane, or an alcohol such as isopropyl alcohol) or that has notbeen subjected to one (or preferably all) of the following refiningstep. Suitable refining protocols are described in International patentapplication no. PCT/EP01/08902 (the contents of this document and allothers described herein are hereby incorporated by reference). Forexample, the oil can be subjected to one or more refining steps whichcan include acid treatment or degumming, alkali treatment or free fattyacid removal, bleaching or pigment removal, filtration, winterisation(or cooling, for example to remove saturated triglycerides), deodorising(or removal of free fatty acids) and/or polishing (or removal ofoil-insoluble substances). All these refining steps are described ingreater detail in PCT/EP01/08902 and can be applied to the stepsdescribed in the present application mutatis mutandis.

The resulting oil is particularly suitable for nutritional purposes, andcan be added to (human) foods or (animal) feedstuffs. Examples includemilk, infant formula, health drinks, bread and animal feed.

Microbial Cells

The microbial cells (or micro-organisms) used in the present inventioncan be any of those described earlier especially in the sectionconcerning PUFAs and microbial oils. They may comprise, or be able toproduce, a PUFA or microbial oil, and suitably the PUFA oil may beextracted or isolated from the cells. They may be in filamentous form,like fungi or bacteria, or single cells like yeast, algae and bacteria.The cells may comprise micro-organisms that are yeast, fungi, bacteriaor algae. Preferred fungi are of the order Mucorales for example, thefungus may be of the genus Mortierella, Phycomyces, Blakeslea orAspergillus. Preferred fungi of the species Mortierella alpina,Blakeslea trispora and Aspergillus terreus.

As far as yeasts are concerned, these are preferably of the genus Pichia(such as of the species Pichia ciferrii) or Saccharomyces.

Bacteria can be of the genus Propionibacterium.

If the cells are from an algae, this is preferably a denoflagellateand/or belongs to the genus Crypthecodinium. Preferred algae of thespecies Crypthecodinium cohnii.

Heating

This can be performed by heating (the cells) directly or indirectly. Theheating, if direct, may be by passing steam into the fermenter. Anindirect method may use a medium via heat exchangers, either through thewall of the fermenter, or with heating coils, or an external heatexchanger such as a plate heat exchanger.

Usually, pasteurisation will take place in the fermenter vessel in whichfermentation has occurred. However, for some organisms (such asbacteria) it is often preferred to remove the cells from the vesselfirst, and then pasteurise. Pasteurisation may take place before otherprocessing of the organisms, for example drying or granulation.

Pasteurisation will usually kill most, or if not all, of themicro-organisms. Following pasteurisation, at least 95%, 96% or even 98%of the micro-organisms have been killed, that is to say they are notalive,

Acidification

In some cases it is desirable to reduce the risk of growth of thepasteurised cells. One possibility is to acidify the cells with asuitable acid. Thus, in order to prevent the outgrowth of microbialspecies, adjusting the cells to a pH range of 3 to 4 may be desirable.However, broader pH ranges can be employed depending on the cells, andso the pH may be adjusted from 2 to 5, optimally at a range of about 3.3to 3.7.

Acidification of the cells may take place before pasteurisation.However, it is preferably conducted afterwards.

The pH can be adjusted by any suitable means, or by any suitable acid.Preferably this is achieved using phosphoric acid, such as 85%, ordiluted 55% or 33% phosphoric acid.

Peroxide Value (POV)

Preferably the POV of the microbial oil is from 4 to 8 or 12, especiallyfor a crude oil. However, the POV may be no more than 3.0, 2.5 or 2.0.However, much lower POV values can be obtained using the process ofinvention, and these values may be less than 1.5 or less than 1.0.Values less than 0.8 or 0.6 and even less than 0.4. POV can be obtained.Values (from embodiments) ranged from 1.3 (or 0.8) to 0.4. The unit (forPOV) is usually meq/kg.

Anisidine Value (AnV)

This value can give a measure of the aldehyde content. Preferably theanisidine value of the microbial oil is from 5, 6, 7 or 10 to 15, 20 or25, especially for a crude oil. Suitably the AnV no more than 20, forexample no more than 15. It may be no more than 10 or even no more than5. Preferably the POV and/or AnV values refer to a crude rather thanrefined, oil. AnV values (in preferred experiments) ranged from 15 to 5,optionally from 12 to 7.

Crude Versus Refined Oils

Some differences between these two oils are presented below. Each crudeor refined oil may have one or more of the features in the followingTable as for the crude or refined oil, as appropriate. A crude oil willusually contain an antioxidant (e.g. tocopherol, ascorbyl palmitate).

Preferred Crude Substance (for crude) oil Refined oil Unsapon- ≤3.5%(w/w) 2.5% (w/w) 1.8 (w/w) ifiables Solvent (e.g. <2000 ppm 100-2000 ppmUndetectable hexane) or ≤1 ppm Phospho- 2-3.5 0.05 lipids % Free fatty  <1% 0.2%  0.08% acids, as oleic POV ≤10 meq/kg 6 meq/kg 1.4 meq/kgNon-solubles <0.5% 0.1% — Phosphorus <1000 mg/kg 5 mg/kg — Silicon <500ppm 100 ppm 24 ppm Arsenic <0.5 mg/kg <0.04 mg/kg <0.5 mg/kg Cadmium<0.2 mg/kg <0.02 mg/kg <0.1 mg/kg Mercury <0.04 mg/kg <0.4 mg/kg <0.04mg/kg Lead <0.1 mg/kg <0.1 mg/kg <0.1 mg/kg Copper <0.2 mg/kg <0.2 mg/kg<0.02 mg/kg Moisture and <1.0% 0.5  <0.02% volatiles Phosphatide 50-100<10    (P/ppm)

Suitably, the crude oil in the present invention may have one or more ofthe following features:

-   -   (a) an unsaponifiables content of from 2.0 to 3.5% (w/w);    -   (b) a solvent (eg. hexane) content of from 10, 50 or 100 ppm up        to 1000, 1500 or 2000 ppm;    -   (c) a free fatty acid content of from 0.1 or 0.2% to 1%, eg.        0.2-0.6 or 0.3-0.5%;    -   (d) a POV value of from 2, 3, 4 or 6 to 10;    -   (e) a phosphorus content of at least 2, 3 or 5 mg/kg;    -   (f) a silicon content of from 50 or 100 ppm to 500 ppm; and/or    -   (g) a water content of less than 1% or from 0.5 to 1 or 2%.        Uses of Oils and PUFAs

A sixth aspect of the invention relates to a composition comprising theoil of the fifth aspect, and where appropriate are or more (additional)substances. The composition may be a foodstuff and/or a food supplementfor animals or humans. In embodiments of the invention which are forhuman consumption the oils may be rendered suitable for humanconsumption, typically by refining or purification of the oil obtainedfrom the microbes.

The composition may be an infant formula or (human) foodstuff. Here thecomposition of the formula may be adjusted so it has a similar amount oflipids or PUFAs to nominal breast milk. This may involve blending themicrobial oil of the invention with other oils in order to attain theappropriate composition.

The composition may be an animal or marine feed composition orsupplement. Such feeds and supplements may be given to any farm animals,in particular sheep, cattle and poultry. In addition, the feeds orsupplements may be given to farmed marine organisms such as fish andshell fish. The composition may thus include one or more feed substancesor ingredients for such an animal.

The oil of the invention may be sold directly as oil and contained inappropriate packaging, typically one piece aluminium bottles internallycoated with epoxy phenolic lacquer, and flushed with nitrogen. The oilmay contain one or more antioxidants (e.g. tocopherol, vitamin E,palmitate) each for example at a concentration of from 50 to 800 ppm,such as 100 to 700 ppm.

Suitable compositions can include pharmaceutical or veterinarycompositions, e.g. to be taken orally or cosmetic compositions. The oilmay be taken as such, or it may be encapsulated, for example in a shell,and may thus be in the form of capsules. The shell or capsules maycomprise gelatine and/or glycerol. The composition may contain otheringredients, for example flavourings (e.g. lemon or lime flavour) or apharmaceutically or veterinary acceptable carrier or excipient.

Preferred features and characteristics of one aspect of the inventionaxe applicable to another aspect mutatis mutandis.

The invention will now be described, by way of example with reference tothe following Examples, which are provided by way of illustration andare not intended to limit the scope.

Example 1

Oxidation during production of a microbial containing PUPA oil isthought to be caused by enzymatic activity, Pasteurisation wasconsidered as a method of stabilising oxidation during processing of themicrobial cells to obtain the microbial oil. The extent of thestabilisation was found to be dependent upon the pasteurisationconditions.

A number of experiments were therefore performed in order to determinewhich pasteurisation conditions could affect the level of oxidation, andin particular the peroxide value (POV) of the oil. Peroxide values weredetermined using the standard protocol detailed in AOCS:Cd8-53.

The experiments follow the following protocol: fermentation; storage;pasteurisation; (microbial oil) extraction; analysis of oil.

The fungus Mortierella alpina was cultivated in a fermenter. Thefermentation lasted approximately 148 hours. M. alpina produced the PUFAcalled arachidonic acid (ARA). The biomass was removed from thefermenter, and stored (at a temperature below −18° C.).

Samples of the M. alpina biomass were removed from the fermentationbroth, while still resident, inside the fermenter, and frozenimmediately.

Various pasteurisation protocols were tried. Pasteurisation wasconducted at three different temperatures, namely 40, 70 and 85° C., Theprotocol followed a three stage process, with a first stage of rapidheating, followed by a plateau (a second or middle stage) at the desiredtemperature, which was the maximum temperature used. There was then arapid cooling (third) stage. Different samples of the biomass weresubjected to a middle (plateau) stage of three different times, namelyone, two and 24 hours.

Following pasteurisation, the microbial oil was obtained using a wetextraction technique. This sample of biomass was filtered, squeezed(under pressure) and the oil extracted.

The microbial oil was then analysed, primarily for the peroxide value(POV) using an AOCS method. The ARA content for some of the samples wasdetermined. Analyses showed that the microbial oil obtained hadapproximately 420 g ARA per kg.

Detailed Protocol: Fermentation and Sample Extraction

One litre of fermentation broth was removed from the fermenter vesseland filtered (Seitz two litre filter, F-FA10). The resulting cake wasthen washed with 600 ml of demineralised water. The wet cake was blowdried for one minute, and then squeezed (using a HAFICO™ apparatus,tincture press, C-OAO21, 300-400 Atm) at 400 bar. The wet extrudate wasthen used to extract a microbial oil with 500 ml of hexane (Merck) atroom temperature (20 to 25° C.) for one hour using an Ultra Turrax™machine. The hexane was then decanted. The remaining cake was thenwashed with 250 ml of fresh hexane (with stirring, for 30 minutes) atroom temperature. The hexane was decanted and then added to thepreviously extracted hexane.

The extract was then filtered using a glass filter in combination with aGFA glass filter. The hexane was then evaporated, using a Rotavapor™machine, from the clear extract at about 50° C. for about 15 minutes.The oil was then transferred into gas-tight cups, and each sample cupwas then flushed with nitrogen for 30 seconds. The sample cup was thenclosed and stored at −18° C.

Pasteurisation Protocols

Three different protocols (A, B and C) were tested. Each was composed ofthree stages, a first heating stage, a second plateau stage (at amaximum temperature) and a third cooling stage. Table 1 below shows theprotocols of the three pasteurisation profiles.

TABLE 1 Combined Area Temp Temp Area Heating/ 40-70- under Time (T, °C.) change Time per under Cooling Time to pass 40° C. t versus T (t, atTime in stage stage profile rate through 40-70° C. times graph minutes)(t) Stage (° C.) (mins) (° C.min) (° C./min) (min) (min) (° C.min)Profile A 0 25 75 70 heat 45 t heat = 75 1687.5 0.6 50 7575 135 70pasteurise 0 t past = 60 4200 0 210 25 cool 45 t cool = 1687.5 0.6 50100 75 Profile B 0 25 (outside 102 72 heat 48 t heat = 4896 0.46 65.1113968 invention for 102 comparison) 162 72 pasteurise 0 t past = 60 43200 360 28 cool 48 t cool = 4752 0.22 135 200.11 198 Profile C 0 7 25 70heat 63 t heat = 25 787.5 2.52 11.90 5607.5 85 70 pasteurise 0 t past =60 4200 0 105 8 cool 62 t cool = 620 3.10 9.68 21.58 20

The three pasteurisation profiles A, B and C are additional showngraphically in FIG. 1. As realised, the area under the temperature (T, °C.) versus time (t, minutes) graph can be calculated for each of thethree steps in each profile, and then summed to give the total areaunder the graph for each of the three profiles. These calculations areadditionally shown in Table 1 above.

The peroxide value (POV) was determined for the oils resulting fromextraction from cells following the three pasteurisation protocols A, Band C. The POV of the extracted oils were 8.7, 14.3 and 2.4respectively. Profile B had slow heating and slow cooling rates and ispresented for comparison only. It gave the highest POV of 14.3.

By contrast, profiles A and C are both within the invention. Profile Ahas a faster heating and cooling rate in the first and third stagesthan, profile B. Preferably, in the invention, the heating and coolingrates are at least as fast as those shown in profile A. Profile A gave aPOV of 8.7.

However, best results were obtained using profile C, which had a POV ofonly 2.4. As can be seen from FIG. 1, this had a very rapid heatingstage, and fast cooling (third) stage.

Example 2

Experiments similar to Example 1 were conducted, except this time thetemperature of pasteurisation was varied more widely, namely at 40° C.(for comparison), 70° C. and 85° C. The profile of temperature (° C.)vs. time (minutes) is shown in FIG. 2 and in Table 2 below. The profilewas essentially the same for all samples, but of course with anextension of the pasteurisation plateau (from one hour to 4 or 24 hours)as appropriate.

TABLE 2 40° C. 70° C. 85° C. Time temp time Temp Time temp 0 10.0 0 8.00 7.0 10 27.0 10 55.0 10 40.0 20 40.1 17 70.0 20 66.0 25 40.0 77 68.2 3079.0 40 41.4 82 44.3 40 83.5 50 41.0 87 31.3 100 79.8 80 38.7 92 21.8105 55.3 85 27.5 97 16.0 110 38.7 90 19.3 102 9.7 115 26.3 95 14.5 12021.0 100 9.7 125 15.2 110 7.5 130 11.3

Samples from two different fermentations (both of M. alpina), ofdifferent length, were tested. Sample nos. 11 to 20. Table 3 had aslightly longer fermentation where about 2 m³ of broth was transferredto an inoculum fermenter and the fermentation extended for 48 hourswithout any further addition of glucose.

After pasteurisation, the samples were processed, starting withfiltration at a pressure of about 1 bar of nitrogen. The resulting cakewas then washed with process water (about 0.6 of the initial brothvolume). De-watering was accomplished using a fruit press at 300 to 400bar piston pressure. Then, 500 ml of fresh hexane was added, and mixedfor one minute using an Ultra-turrax machine for one minute. Extractionthen took place for about one hour at ambient temperature. Followingfiltration, the resulting cake was washed With 250 ml of fresh hexane,and the resulting solvent was evaporated under vacuum at 60 to 70° C.,It was then flushed with nitrogen and stored at −18° C.

The results are shown in Table 3, which includes the first and secondmeasured peroxide values, and an average of these two values, as well asthe anisidine value (AnV). The reduction in POV and AnV are also shownin FIGS. 3 and 4 (for the shorter and longer fermentations,respectively).

TABLE 3 Sample No. T_(past) (° C.) t_(past) (hrs) POV1 POV2 POV_(ave)AnV 1 — 0 6.0 5.4 5.7 25.7 2 40 1 8.8 8.5 8.6 25.9 3 40 4 3.8 3.8 3.827.1 4 40 24 2.1 2.2 2.1 21.0 5 70 1 2.2 2.2 30.2 6 70 3.5 1.1 1.1 1.133.5 7 70 22 0.7 0.7 1.0 15.9 8 85 1 1.2 1.2 1.2 25.9 9 85 3.3 0.7 0.80.7 27.1 10 85 20.5 0.5 0.5 0.5 12.9 11 — 0 5.9 5.4 5.6 39.3 12 40 1 9.910.1 10.0 38.7 13 40 4 4.8 4.5 4.6 40.7 14 40 24 2.5 3.0 2.8 32.3 15 701 2.7 2.8 2.7 40.3 16 70 3.5 1.6 1.7 1.3 32.7 17 70 22 1.0 0.9 1.3 14.518 85 1 1.8 1.8 1.8 39.7 19 85 3.3 1.1 1.1 1.1 32.4 20 85 20.5 0.9 1.00.9 16.1

From the results it will be seen that with no pasteurisation, the POVwas 5.6 or 5.7. Pasteurisation at 40° C. did reduce the POV, but arelatively long time (such as 24 hours) at the pasteurisationtemperature was required in order to reduce the POV to an acceptablevalue of 2.1.

Higher temperatures were considerably more successful. For example,pasteurisation for only 1 hour at 70° C. gave a POV of 2.2, whencompared to a POV of 2.1 for 24 hours at 40° C. Even better values wereobtained at higher temperatures, with 85° C. for 1 hour giving a POVvalue of only 1.2. (These figures are quoted for the shorterfermentation, although similar results can be found with cells grown inthe longer fermentation).

FIGS. 3 and 4 thus show graphically how the POV and AnV values changewith respect to different pasteurisation times. As expected, longerpasteurisation times give lower AnV and POV values. However, of moreimportance is the use of relatively high temperatures duringpasteurisation. A marked decrease in AnV and POV was found when thepasteurisation temperature (T_(past)) was increased to 70° C., and evenlower values were found at 85° C., (The top three lines, indicated bycrosses, filled circles and asterisks show the AnV values, while thelower three lines, indicated by diamonds, squares and triangles, givethe POV values).

Table 4 below shows the calculated product tT (in ° C. minute) for thenine different pasteurisation protocols (three different plateautemperatures and for three different times). This product in effectrepresents the area under the graph (of time, t, minutes vs.temperature, T, ° C.) for the plateau stage (after the heating stage butbefore the cooling stage).

TABLE 4 Temp (T, ° C.) Time (t, hrs/mins) 40 70 85 1 (60)  2,400 4,2005,100 4 (240) 9,600 16,800 20,400 24 (1440) 57,600 100,800 122,400

Example 3

Further pasteurisation trials were conducted using fermentation broth,following fermentation on a production scale, using the fungus M.alpina, as previously exemplified. Unpasteurised broth (800 litres) wastransported, and stored at 4° C. The broth was then transferred to astirred vessel of 700 litres and 10 different pasteurisation protocolsperformed.

Firstly, pasteurisation was conducted at five different (maximum)temperatures, namely 140, 120, 100, 80 and 60° C. with a residence(plateau) time (at maximum temperature) of 8 seconds, Secondly,pasteurisation was conducted at 140, 120, 100, 80 and 60° C. with aresident (plateau) time*at maximum temperature of 300 seconds.

Samples (2 litres) were taken and then frozen directly at −18° C.Sterile samples (200 ml) were taken and frozen, and crude ARA oilrecovered from the samples using the following protocol.

A sample of fermentation broth (1.7 litres) was filtered at 1 bar of N₂.The cake was washed with 0.6 volumes of condensed water, and squeezedfor about 5 minutes at 400 kg/cm². Then, n-hexane (500 ml) was added tothe wet cake, and crumbled using an Ultra Turrax machine at 24,000 rpm.The oil was extracted at ambient temperature (about 21° C.) over about110 minutes. The suspension was filtered with a vacuum using a GF/AWhatman filter medium. The cake was washed with 250 ml of fresh hexane.The hexane was evaporated for 15 minutes in a water bath, having atemperature of about 60 to 70° C. The resulting oil was then transferredto gas-tight sample cups, which were flushed with nitrogen for 30seconds, and then closed and stored prior to analysis at −18° C.

FIGS. 5, 6 and 7 provide the data following analysis. FIGS. 6 and 7 showthe time against temperature profiles for the two sets of experiments,firstly of the plateau (residence) time of 8 seconds, and secondly forthe plateau (residence) time of 5 minutes, at each of the 5 temperaturesettings, respectively. As will be seen from the graphs, the horizontalmiddle line (representing 8 seconds or 5 minutes) shows the plateaustage.

FIG. 5 shows the resulting POV and AnV values for all 10 ofpasteurisation regimes. As will be seen, lower POV values were obtainedwith increasingly higher temperatures, and the longer residence time (5minutes) gave the lowest POV value.

The invention claimed is:
 1. A microbial oil comprising at least 40% ofarachidonic acid (ARA); a peroxide value (POV) of less than 3.0; and ananisidine value (AnV) of no more than
 25. 2. The microbial oil of claim1 wherein the oil is a crude or unrefined oil.
 3. The oil according toclaim 1, wherein the oil has a triglyceride content of at least 90%. 4.The oil according to claim 1, wherein the oil comprises at least 45% ofARA.
 5. The oil according to claim 1, wherein the oil has an AnV of nomore than
 20. 6. The oil according to claim 1, wherein the oil has anAnV of no more than
 15. 7. The oil according to claim 1, wherein the oilhas an AnV of from 5 to
 25. 8. The oil according to claim 1, wherein theoil has an AnV of from 5 to
 20. 9. The oil according to claim 1, whereinthe oil has an AnV of from 5 to
 15. 10. The oil according to claim 1,wherein the oil has an AnV of from 10 to
 20. 11. The oil according toclaim 1, wherein the oil has a POV of no more than 2.5 or 2.0.
 12. Theoil according to claim 1, wherein the oil has a POV of less than 1.5 orless than 1.0.
 13. The oil according to claim 1, wherein the oil has aPOV from 0.4 to 3.0, from 0.4 to 2.5, from 0.4 to 2.0, from 0.4 to 1.5,from 0.4 to 1.3, from 0.4 to 1.0, or from 0.4 to 0.8.
 14. The oilaccording to claim 1, wherein the oil is extracted or isolated fromcells which are fungi.
 15. The oil according to claim 14, wherein thecells are of the genus Mortierella.
 16. The oil according to claim 15,wherein the cells are of the species Mortierella alpina.
 17. The oilaccording to claim 1, wherein the oil is subjected to one or morerefining steps.
 18. The oil of claim 17, wherein the refining stepsinclude acid treatment or degumming, alkali treatment or free fatty acidremoval, bleaching or pigment removal, filtration, winterization,deodorizing and/or polishing.
 19. The oil according to claim 17 or 18,wherein the refined oil is added to human foods or animal foodstuffs.20. The oil according to claim 17 or 18, wherein the refined oil isadded to infant formula.
 21. The microbial oil of claim 1 obtained by aprocess for pasteurising microbial cells or microorganism that comprisesthree stages, the process comprising: (a) a (first) heating stage, (b) a(second) plateau stage at which the cells are maintained at a constanttemperature and (c) a (third) cooling stage, and further comprisingextracting or isolating the oil from the pasteurized cells ormicroorganisms.
 22. The microbial oil of claim 1 obtained by a processof pasteurising microbial cells or microorganisms, the processcomprising: (a) heating the cells or microorganisms from 40° C. to 60°C. in no more than 30 minutes or (b) heating the cells or microorganismsfrom 40° C. to 60° C. at a rate greater than 0.5° C./minute or (c)heating the cells or microorganisms from 40° C. to 60° C. in no morethan 30 minutes at a rate greater than 0.5° C./minute or (d) heating thecells or microorganisms and maintaining the heated cells ormicroorganisms at an elevated temperature (T, ° C.) for a time (t,minutes) at a plateau stage, wherein the product T.t is from 140 to100,800° C. minute.
 23. The oil according to claim 21 or claim 22,wherein the oil has an AnV of no more than
 20. 24. The oil according toclaim 21 or claim 22, wherein the oil has an AnV of no more than
 15. 25.The oil according to claim 21 or claim 22, wherein the oil has an AnV offrom 5 to
 25. 26. The oil according to claim 21 or claim 22, wherein theoil has an AnV of from 5 to
 20. 27. The oil according to claim 21 orclaim 22, wherein the oil has an AnV of from 5 to
 15. 28. The oilaccording to claim 21 or claim 22, wherein the oil has an AnV of from 10to
 20. 29. The oil according to claim 21 or claim 22, wherein the oilhas a POV of no more than 2.5 or 2.0.
 30. The oil according to claim 21or claim 22, wherein the oil has a POV of less than 1.5 or less than1.0.
 31. The oil according to claim 21 or claim 22, wherein the oil hasa POV from 0.4 to 3.0, from 0.4 to 2.5, from 0.4 to 2.0, from 0.4 to1.5, from 0.4 to 1.3, from 0.4 to 1.0, or from 0.4 to 0.8.